Protocol:
The protocol includes four main steps: buffer set up, lysis, debris
removal, and resuspension.
Buffer Set-Up
Table of Buffers:
#install.packages("openxlsx")
#install.packages("here")
library(openxlsx)
library(here)
## here() starts at /Users/debs/Nelson Lab
library(openxlsx)
library(dplyr)
##
## Attaching package: 'dplyr'
## The following objects are masked from 'package:stats':
##
## filter, lag
## The following objects are masked from 'package:base':
##
## intersect, setdiff, setequal, union
library(knitr)
library(kableExtra)
##
## Attaching package: 'kableExtra'
## The following object is masked from 'package:dplyr':
##
## group_rows
library(here)
# Read the data
df <- read.xlsx(here("buffers.xlsx"))
url1 <- df$url1
df2 <- df %>% select(-url1) %>%
mutate_all(~ replace(., is.na(.), ""))
## Warning: Using an external vector in selections was deprecated in tidyselect 1.1.0.
## ℹ Please use `all_of()` or `any_of()` instead.
## # Was:
## data %>% select(url1)
##
## # Now:
## data %>% select(all_of(url1))
##
## See <https://tidyselect.r-lib.org/reference/faq-external-vector.html>.
## This warning is displayed once every 8 hours.
## Call `lifecycle::last_lifecycle_warnings()` to see where this warning was
## generated.
df3 <- df2 %>%
kbl(booktabs = T, align = "c") %>%
kable_styling() %>%
column_spec(1, bold = T, color = "#990000") %>%
column_spec(1:ncol(df2), width = "12em") %>% # Set all columns to same width
row_spec(1:nrow(df2), align = "c" ) %>% # Bold the top row (header) with red color
row_spec(3:nrow(df2), align = "c") # Align the rest of the rows to center
df3
|
Buffer.Name
|
Solute/.Component.1
|
Solvent/.Component.2
|
Solvent/.Compound.3
|
|
1X PBS
|
10 mL of 10X PBXS
|
90 mL DI water
|
N/A
|
|
10% BSA
|
10 g BSA powder
|
100 mL DI water
|
N/A
|
|
Lysis Buffer
|
Reducing Agent B (PN:2000558)
|
Surfactant A (PN: 2000559)
|
Lysis Reagent (PN: 2000558)
|
|
Debris Buffer
|
Reducing Agent B (PN:2000558)
|
N/A
|
Debris Removal Reagent (PN: 2000560)
|
|
Wash and Resuspention Buffer
|
1X PBS
|
10% BSA
|
Rnase Inhbitor (PN: 2000565)
|
Start with the Wash and Resuspension Buffer, which is composed of the
1X PBS and 10% BSA solutions that are not included in the kit.
Generally, make a stock of 100 mL of each and keep chilled at 4 degrees
Celcius. Refer to the table above and the instructions for components
and amounts.
1X PBS: Dilute 10 mL into 90 mL of Ultra-Pure Water. Invert to mix
thoroughly.
10% BSA: 10 grams of powder BSA to 100 mL of Ultra-Pure Water. Mix
gently by swirling to prevent excess frothing.
Next, retrieve all components of the 10X Nuclei Isolation Kit, which
should include the Lysis Reagent, Debris Removal Reagent, Reducing Agent
B, Surfactant A, and RNAse Inhibitor. Also retrieve the Sample
Dissociation Tube from the room temperature Consumables portion.
Place everything except for the Reducing Agent B on ice and begin to
prepare the buffers. After Reducing Agent B thaws and everything is
briefly vortexed, begin preparing the buffers.
Per reaction, the Lysis Buffer requires 550 µl Lysis reagent, 0.55 µl
Reducing Agent B, and 5.5 µl Surfactant A. Per reaction, the Debris
Removal Buffer requires 550 µl Debris Removal Reagent and 0.55 µl of the
Reducing Agent B. Per reaction, the Wash and Resuspension Buffer
requires 2,887.5 µl 1X PBS, 330 µl 10% BSA, and 82.5 µl of RNase
Inhibitor for sequencing.
Keep all completed buffers labeled and on ice. Pre-chill the
centrifuge to 4 degrees C.
Main Procedure
Next, retrieve flash frozen half kidney from the -80 degrees storage.
While frozen, use tweezers or scissors to remove the kideny and place it
in a pre-chilled Sample Dissociation Tube. Add 200 µl of the Lysis
Buffer and wait for the kidney to mostly thaw. Using sterile scissors,
chop up the kidney into small pieces, closing and opening scissors at
least 50 times.
Then using the green pestle provided in the room-temperature pack,
grind up the tissue, pressing down and twisting at least 20 times or
until the solution is smooth with no visible chunks.
Next, add 300 µl of the Lysis buffer and pipette up and down, making
sure that there are no chunks that block the pipette tip. Incubate the
solution on ice for 8 minutes, starting the timer after completing the
grinding step.
Next, pipette the dissociated tissue solution onto the Nuclei
Isolation Column in the collection Tube (both included in room
temperature Consummables) and spin in the centrifuge for 16,000 rcf, 20
sec, at 4 degrees C. Make sure that this cycle is evenly balanced in the
centrifuge, because of the high speed.
After the cycle ends, discard the column and vortex the flow-through
for 10 seconds to resuspend the nuclei. Then spin in the centrifuge at
700 rcf, 3 min, 4 degrees C. Make sure that when placing the Nuclei
Isolation Column, position it within the centrifuge so that the hinge of
the lid points out, the front tab facing in. This will make it easier to
find the pellet and avoid losing nuclei.
Next, remove the supernatent, being careful to avoid the pellet and
angling the pipette tip away from the hinge side of the column.
Resuspend the pellet with 500 µl of the Debris Removal Buffer. Spin at
700 rcf for 10 minutes at 4 degrees C. While waiting, mix the final
Resuspension Buffer, which should be 0.04% BSA in PBS.
Remove the supernatant again and resuspend in 1 mL of the Wash and
Resuspension Buffer. Spin at 500 rcf for 5 minutes, 4 degrees C. Remove
supernatant, resuspend again in 1 mL of Wash and Resuspension Buffer.
Spin again at 500 rcf for 5 minutes, 4 degrees C.
Finally, resuspend the. nuclei pellet in 500 µl of the Final Wash and
Resuspension Buffer (0.04% BSA). Vortex the solution for 3 seconds and
let it sit undisturbed for 15 minutes.
Now, depending on QC or following uses, use the concentrated
solution.
Nuclei Count with
Light Microscope:
With the nuclei solution, add 2 µl to 18 µl of Trypan Blue, pipetting
up and down into the TB at least 10 times to mix thoroughly. Note that
while Trypan Blue does not aggregate in whole cells, the compound does
aggregate within the exposed nuclei, which is what we will be checking
for. Add 10 µl to one of the wells on a hemocytometer, making sure the
solution fills the space. Observe on a light microscope, counting to get
an approximation and assessment of nuclei quality.
Microscope used in this procedure:
link
to hemocytometer counter app
From this procedure, the average expected nuclei concentration count
is: 500 nuclei per microliter This is approximately 50 to 60 nuclei per
quadrant in the hemocytometer.
Nuclei Quality
Check:
Priority Check:
No clumps, with an even distribution
Quality:
A grade Nuclei: Good, circular shape with no blebbing, halo of Trypan
Blue around the edges that suggest an undisturbed nuclear envelope. No
aggregation.
B grade Nuclei: Slightly irregular shapes, still has the halo but
less bright or more uneven
C grade Nuclei: Losing more of normal shape, small blebbing, paler
color
D/F grade Nuclei: Complete loss of structure, no blue halo,
aggregates
Example of a graded sample:

Example of grading with reasoning. Note: this is an image of an
aggregate that was included because of a wide variety of quality, but as
a general rule aggregates indicate a low quality overall sample.

Example of a sample viewed at 10x and 40x, grading the individual
nuclei quality is easier at 40x but it is recommended to conduct overall
sample quality checks at 10x magnification.
